We are examining the role of thrombospondin-1 (TSP1) in regulating tumor growth, angiogenesis, and metastasis. These studies examine the direct effects of TSP1 expression on tumor cells, its role in neovascularization of tumors, and its role in tumor cell interactions with endothelium and the host immune response during metastasis. TSP1 is a matricellular protein that both stimulates and inhibits specific cellular responses. Some of the diversity in cellular responses to TSP1 may be explained by differential expression of several TSP1 receptors that induce distinct signals. However, we have also found that a single TSP1 receptor, the a3b1 integrin, can mediate cell type-specific responses to TSP1 through differential regulation of its activation state. The a3b1 integrin is the major mediator of breast carcinoma cell adhesion and chemotaxis to TSP1. This integrin is constitutively expressed, but it is normally inactive in these cells. Signaling through G proteins, IGF1, and CD98 specifically activate the a3b1 integrin in breast carcinoma cells. This regulation is tumor specific, since EGF but not IGF1 stimulates function of a3b1 integrin in SCLC cells. The a3b1 integrin is also expressed in endothelial cells and regulates angiogenesis, but its activity to recognize TSP1 is controlled through VE-cadherin signaling rather than the growth factors that regulate its function in tumor cells. We now report that conformational regulation of TSP1 can also modulate its binding to this integrin. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was specifically stimulated when TSP1 bound to fibronectin. Binding of the fibronectin/TSP1 complex to melanoma cells was mediated by a4b1 and a5b1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1. This enhancement of adhesion was mediated by a3b1 integrin and required that the a3b1 integrin be in an active state. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by a3b1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction. These studies have provided new insights into the mechanisms by which TSP1 expression inhibits tumor growth and neovascularization. Using microarray expression analysis, we recently demonstrated that TSP1 acts globally to inhibit TCR-mediated T lymphocyte activation. Using CD69 expression as an indicator of T cell activation, TSP1 was shown to antagonize TCR and CD47-mediated T cell activation but not activation stimulated by protein kinase C. This inhibitory activity required two TSP1 receptors: CD47 and heparan sulfate proteoglycan. Signals from these receptors inhibited TCR signaling downstream of ZAP70 and LAT but upstream of NF-AT. Therefore, expression of TSP1 in tumors or tumor stroma may limit T cell activation at these sites. Using synthetic peptides and recombinant fragments, we have identified functional sites in thrombospondin-1 that express these activities. Peptide sequences have been identified that interact specifically with heparan sulfate proteoglycans, a3b1 integrin, and a4b1 integrin. Stable analogs of these peptides were prepared using D-reverse peptides and inhibit tumor growth and angiogenesis in animal models. These stable peptide analogs have potential clinical applications in cancer and other diseases associated with abnormal angiogenesis.